Infection and inflammation are leading causes of newborn morbidity and mortality. Many sources of infection including blood-stream infections (sepsis) and necrotizing enterocolitis (NEC) are both common and unique to the neonate as is the systemic response observed in this patient population. NEC is the most common life- threatening gastrointestinal disease among neonates and a leading cause of overall premature infant morbidity and mortality. Further complicating the management of these critically ill infants is the considerable overlap in the early clinical presentation of NEC and Sepsis. Despite decades of research and clinical practice, the approach to the neonate under suspicion of sepsis is non-specific and largely supportive. Illustrating this point is the lack of specific molecular diagnostics for NEC or sepsis. Rather, current practice continues to rely on non-specific laboratory tests to assist in diagnosing disease and monitoring response to therapy (antibiotics etc.). As a result, the identification of neonates with NEC and more importantly progressive forms of the disease by current clinical parameters currently occurs at a time when the course of disease is un-alterable. Similarly, the empiric over-use of antibiotics in the neonatal patient population awaiting culture results for possible sepsis results in potentially significant additional morbidity, including increased incidence of NEC. These observations highlight the increasing biomedical burden imposed by NEC and sepsis and therefore the need for specific molecular disease classifiers in order to assist in altering their onset and progression. To address this significant unmet need, we have initiated protein biomarker studies in the blood and urine of neonates that are both diagnostic and prognostic of NEC and sepsis. In this proposal, we intend to verify and validate an optimized panel of biomarkers that can then be utilized as a disease specific molecular test for neonatal critical care. It is hypothesized that dynamic monitoring of disease specific biomarker panels will advance our understanding of the clinical pathogenesis of NEC and sepsis in neonates and lead to more timely and objective patient disease classification and ultimately to improved disease specific treatments. The proposed research plan has three Specific Aims: 1. Verify and validate body fluid candidate protein biomarkers of NEC 2. Discover and verify tissue-specific biomarkers, cross-validate to body fluid panels of biomarkers 3. Conduct independent prospective validation of optimized biomarker panels on clinical samples The PI for this project has assembled a multi-disciplinary team of scientists with complimentary areas of expertise in proteomics, bioinformatics, and related technologies in order to pursue this project and its specific aims. Further empowering these studies is our partnership with the NICHD sponsored Neonatal Research Network (NRN) that will collect the biologic samples and clinical data on infants with a possible diagnosis of either NEC or sepsis. The NRN is a NIH sponsored consortium of 22 leading university-based children's hospitals with clinical investigators and research coordinators that together comprise the needed infrastructure and possess the experience necessary to conduct the proposed studies. The Steering Committee that includes a PI from each of the NRN sites has given their approval for these studies thus validating their potential significant impact on neonatal clinical care.